DESCRIPTION: The nucleotide excision DNA repair (NER) pathway exists as a widely distributed biological mechanisms for removing various structurally unrelated DNA adducts. Preferential NER occurs for some DNA adducts due to a transcription-coupled repair (TCR) mechanisms that is proposed to initiate as consequence of arrested RNA polymerase II (RNA pol II) at transcription blocking lesions. The objective of this proposal is aimed at understanding how DNA repair enzymes recognize the arrested transcription complex and facilitate transcription-coupled repair. Taking advantage of an in vitro transcription assay that utilizes purified rat liver RNA pol II and initiation factors, the P.I.s propose to determine the structural properties of transcription complexe arrested at cyclobutane pyrimidine dimer (CPD) or other lesions. A comparison will be made to the properties of complexes arrested at naturally occurring arrest sites. To achieve the objective, three specific aims were described. First, three parameters of the RNA pol II complex arrested at CPD sites will b examined. The P.I.s have proposed 1) to determine the site of the front and rear edge of the RNA pol II arrested complex on a site-specific CPD-containing DNA ligand; 2) to locate the position of the catalytic site of RNA pol II with respect to the 3'-end of the incomplete transcript following transcription arrest; and 3) to determine the effect of nucleotide sequence context on the footprint of RNA pol II complexes in bent DNA. The second aim centers on examining the unique character of RNA pol II complexes arrested at CPD sites compared to complexes arrested at other types of pausing sites (e.g. sequence dependent arrest sites, DNA binding protein, DNA binding drug and nucleotide depletion). The third aim is proposed to extend the analysis described in Specific Aims 1 and 2 for CPDs to other types of DNA lesions (psoralen crosslinks, psoralen monoadducts, thymine glycol, abasic sites, and single-strand breaks). It is hoped that an analysis of different types of arrested RNA pol II complexes will provide an understanding of the recognition process used to attract DNA repair enzymes and initiate TCR.